Laurie C. Van De Werfhorst

Performance of forty-one microbial source tracking methods: a twenty-seven lab evaluation study.

The last decade has seen development of numerous new microbial source tracking (MST) methodologies, but many of these have been tested in just a few laboratories with a limited number of fecal samples. This method evaluation study examined the specificity and sensitivity of 41 MST methodologies by analyzing data generated in 27 laboratories. MST methodologies that targeted human, cow, ruminant, dog, gull, pig, horse, and sheep were tested against sewage, septage, human, cow, dog, deer, pig, chicken, pigeon, gull, horse, and goose fecal samples. Each laboratory received 64 blind samples containing a single source (singletons) or two sources (doubletons), as well as diluted singleton samples to assess method sensitivity. Laboratories utilized their own protocols when performing the methods and data were deposited in a central database before samples were unblinded. Between one and seven laboratories tested each method. The most sensitive and specific assays, based on an analysis of presence/absence of each marker in target and non-target fecal samples, were HF183 endpoint and HF183SYBR (human), CF193 and Rum2Bac (ruminant), CowM2 and CowM3 (cow), BacCan (dog), Gull2SYBR and LeeSeaGull (gull), PF163 and pigmtDNA (pig), HoF597 (horse), PhyloChip (pig, horse, chicken, deer), Universal 16S TRFLP (deer), and Bacteroidales 16S TRFLP (pig, horse, chicken, deer); all had sensitivity and specificity higher than 80% in all or the majority of laboratories. When the abundance of MST markers in target and non-target fecal samples was examined, some assays that performed well in the binary analysis were found to not be sensitive enough as median concentrations fell below a minimum abundance criterion (set at 50 copies per colony forming units of enterococci) in target fecal samples. Similarly, some assays that cross-reacted with non-target fecal sources in the binary analysis were found to perform well in a quantitative analysis because the cross-reaction occurred at very low levels. Based on a quantitative analysis, the best performing methods were HF183Taqman and BacH (human), Rum2Bac and BacR (ruminant), LeeSeaGull (gull), and Pig2Bac (pig); no cow or dog-specific assay met the quantitative specificity and sensitivity criteria. Some of the best performing assays in the study were run by just one laboratory so further testing of assay portability is needed. While this study evaluated the marker performance in defined samples, further field testing as well as development of frameworks for fecal source allocation and risk assessment are needed.

Diversity, Composition, and Geographical Distribution of Microbial Communities in California Salt Marsh Sediments

ABSTRACT The Pacific Estuarine Ecosystem Indicators Research Consortium seeks to develop bioindicators of toxicant-induced stress and bioavailability for wetland biota. Within this framework, the effects of environmental and pollutant variables on microbial communities were studied at different spatial scales over a 2-year period. Six salt marshes along the California coastline were characterized using phospholipid fatty acid (PLFA) analysis and terminal restriction fragment length polymorphism (TRFLP) analysis. Additionally, 27 metals, six currently used pesticides, total polychlorinated biphenyls and polycyclic aromatic hydrocarbons, chlordanes, nonachlors, dichlorodiphenyldichloroethane, and dichlorodiphenyldichloroethylene were analyzed. Sampling was performed over large (between salt marshes), medium (stations within a marsh), and small (different channel depths) spatial scales. Regression and ordination analysis suggested that the spatial variation in microbial communities exceeded the variation attributable to pollutants. PLFA analysis and TRFLP canonical correspondence analysis (CCA) explained 74 and 43% of the variation, respectively, and both methods attributed 34% of the variation to tidal cycles, marsh, year, and latitude. After accounting for spatial variation using partial CCA, we found that metals had a greater effect on microbial community composition than organic pollutants had. Organic carbon and nitrogen contents were positively correlated with PLFA biomass, whereas total metal concentrations were positively correlated with biomass and diversity. Higher concentrations of heavy metals were negatively correlated with branched PLFAs and positively correlated with methyl- and cyclo-substituted PLFAs. The strong relationships observed between pollutant concentrations and some of the microbial indicators indicated the potential for using microbial community analyses in assessments of the ecosystem health of salt marshes.

Performance of human fecal anaerobe-associated PCR-based assays in a multi-laboratory method evaluation study.

A number of PCR-based methods for detecting human fecal material in environmental waters have been developed over the past decade, but these methods have rarely received independent comparative testing in large multi-laboratory studies. Here, we evaluated ten of these methods (BacH, BacHum-UCD, Bacteroides thetaiotaomicron (BtH), BsteriF1, gyrB, HF183 endpoint, HF183 SYBR, HF183 Taqman(®), HumM2, and Methanobrevibacter smithii nifH (Mnif)) using 64 blind samples prepared in one laboratory. The blind samples contained either one or two fecal sources from human, wastewater or non-human sources. The assay results were assessed for presence/absence of the human markers and also quantitatively while varying the following: 1) classification of samples that were detected but not quantifiable (DNQ) as positive or negative; 2) reference fecal sample concentration unit of measure (such as culturable indicator bacteria, wet mass, total DNA, etc); and 3) human fecal source type (stool, sewage or septage). Assay performance using presence/absence metrics was found to depend on the classification of DNQ samples. The assays that performed best quantitatively varied based on the fecal concentration unit of measure and laboratory protocol. All methods were consistently more sensitive to human stools compared to sewage or septage in both the presence/absence and quantitative analysis. Overall, HF183 Taqman(®) was found to be the most effective marker of human fecal contamination in this California-based study.

Sewage Exfiltration As a Source of Storm Drain Contamination during Dry Weather in Urban Watersheds

Separating storm drains and sanitary sewers is expected to control sewage pollution, for example, from combined sewer overflows, and to reduce excessive stormwater flow to wastewater treatment plants. However, sewage contamination has been found in such separated storm drain systems in urban areas during dry-weather flow. To determine whether transmission of sewage is occurring from leaking sanitary sewers directly to leaking separated storm drains, field experiments were performed in three watersheds in Santa Barbara, CA. Areas with high and low risks for sewage exfiltration into storm drains were identified, and rhodamine WT (RWT) dye pulses were added to the sanitary sewers. RWT was monitored in nearby storm drain manholes using optical probes set up for unattended continuous monitoring. Above-background RWT peaks were detected in storm drains in high-risk areas, and multiple locations of sewage contamination were found. Sewage contamination during the field studies was confirmed using the human-specific Bacteroidales HF183 and Methanobrevibacter smithii nifH DNA markers. This study is the first to provide direct evidence that leaking sanitary sewers can directly contaminate nearby leaking storm drains with untreated sewage during dry weather and suggests that chronic sanitary sewer leakage contributes to downstream fecal contamination of coastal beaches.

Characterization of Coastal Urban Watershed Bacterial Communities Leads to Alternative Community-Based Indicators

Background Microbial communities in aquatic environments are spatially and temporally dynamic due to environmental fluctuations and varied external input sources. A large percentage of the urban watersheds in the United States are affected by fecal pollution, including human pathogens, thus warranting comprehensive monitoring. Methodology/Principal Findings Using a high-density microarray (PhyloChip), we examined water column bacterial community DNA extracted from two connecting urban watersheds, elucidating variable and stable bacterial subpopulations over a 3-day period and community composition profiles that were distinct to fecal and non-fecal sources. Two approaches were used for indication of fecal influence. The first approach utilized similarity of 503 operational taxonomic units (OTUs) common to all fecal samples analyzed in this study with the watershed samples as an index of fecal pollution. A majority of the 503 OTUs were found in the phyla Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria. The second approach incorporated relative richness of 4 bacterial classes (Bacilli, Bacteroidetes, Clostridia and α-proteobacteria) found to have the highest variance in fecal and non-fecal samples. The ratio of these 4 classes (BBC∶A) from the watershed samples demonstrated a trend where bacterial communities from gut and sewage sources had higher ratios than from sources not impacted by fecal material. This trend was also observed in the 124 bacterial communities from previously published and unpublished sequencing or PhyloChip- analyzed studies. Conclusions/Significance This study provided a detailed characterization of bacterial community variability during dry weather across a 3-day period in two urban watersheds. The comparative analysis of watershed community composition resulted in alternative community-based indicators that could be useful for assessing ecosystem health.

Potential mechanisms and environmental controls of TiO2 nanoparticle effects on soil bacterial communities.

It has been reported that engineered nanoparticles (ENPs) alter soil bacterial communities, but the underlying mechanisms and environmental controls of such effects remain unknown. Besides direct toxicity, ENPs may indirectly affect soil bacteria by changing soil water availability or other properties. Alternatively, soil water or other environmental factors may mediate ENP effects on soil bacterial communities. To test, we incubated nano-TiO2-amended soils across a range of water potentials for 288 days. Following incubation, the soil water characteristics, organic matter, total carbon, total nitrogen, and respiration upon rewetting (an indicator of bioavailable organic carbon) were measured. Bacterial community shifts were characterized by terminal restriction fragment length polymorphism (T-RFLP). The endpoint soil water holding had been reported previously as not changing with this nano-TiO2 amendment; herein, we also found that some selected soil properties were unaffected by the treatments. However, we found that nano-TiO2 altered the bacterial community composition and reduced diversity. Nano-TiO2-induced community dissimilarities increased but tended to approach a plateau when soils became drier. Taken together, nano-TiO2 effects on soil bacteria appear to be a result of direct toxicity rather than indirectly through nano-TiO2 affecting soil water and organic matter pools. However, such directs effects of nano-TiO2 on soil bacterial communities are mediated by soil water.

Evaluation of molecular community analysis methods for discerning fecal sources and human waste.

Molecular microbial community analyses provide information on thousands of microorganisms simultaneously, and integrate biotic and abiotic perturbations caused by fecal contamination entering water bodies. A few studies have explored community methods as emerging approaches for microbial source tracking (MST), however, an evaluation of the current state of this approach is lacking. Here, we utilized three types of community-based methods with 64 blind, single- or dual-source, challenge samples generated from 12 sources, including: humans (feces), sewage, septage, dogs, pigs, deer, horses, cows, chickens, gulls, pigeons, and geese. Each source was a composite from multiple donors from four representative geographical regions in California. Methods evaluated included terminal restriction fragment polymorphism (TRFLP), phylogenetic microarray (PhyloChip), and next generation (Illumina) sequencing. These methods correctly identified dominant (or sole) sources in over 90% of the challenge samples, and exhibited excellent specificity regardless of source, rarely detecting a source that was not present in the challenge sample. Sensitivity, however, varied with source and community analysis method. All three methods distinguished septage from human feces and sewage, and identified deer and horse with 100% sensitivity and 100% specificity. Method performance improved if the composition of blind dual-source reference samples were defined by DNA contribution of each single source within the mixture, instead of by Enterococcus colony forming units. Data analysis approach also influenced method performance, indicating the need to standardize data interpretation. Overall, results of this study indicate that community analysis methods hold great promise as they may be used to identify any source, and they are particularly useful for sources that currently do not have, and may never have, a source-specific single marker gene.

Soybean Plants Modify Metal Oxide Nanoparticle Effects on Soil Bacterial Communities

Engineered nanoparticles (ENPs) are entering agricultural soils through land application of nanocontaining biosolids and agrochemicals. The potential adverse effects of ENPs have been studied on food crops and soil bacterial communities separately; however, how ENPs will affect the interacting plant-soil system remains unknown. To address this, we assessed ENP effects on soil microbial communities in soybean-planted, versus unplanted, mesocosms exposed to different doses of nano-CeO2 (0-1.0 g kg(-1)) or nano-ZnO (0-0.5 g kg(-1)). Nano-CeO2 did not affect soil bacterial communities in unplanted soils, but 0.1 g kg(-1) nano-CeO2 altered soil bacterial communities in planted soils, indicating that plants interactively promote nano-CeO2 effects in soil, possibly due to belowground C shifts since plant growth was impacted. Nano-ZnO at 0.5 g kg(-1) significantly altered soil bacterial communities, increasing some (e.g., Rhizobium and Sphingomonas) but decreasing other (e.g., Ensifer, Rhodospirillaceae, Clostridium, and Azotobacter) operational taxonomic units (OTUs). Fewer OTUs decreased from nano-ZnO exposure in planted (41) versus unplanted (85) soils, suggesting that plants ameliorate nano-ZnO effects. Taken together, plants--potentially through their effects on belowground biogeochemistry--could either promote (i.e., for the 0.1 g kg(-1) nano-CeO2 treatment) or limit (i.e., for the 0.5 g kg(-1) nano-ZnO treatment) ENP effects on soil bacterial communities.

Microbial Source Tracking in a Coastal California Watershed Reveals Canines as Controllable Sources of Fecal Contamination

Elevated levels of fecal indicator bacteria (FIB), including Escherichia coli and enterococci, trigger coastal beach advisories and signal public health risks. Solving FIB pollution in suburban coastal watersheds is challenging, as there are many potential sources. The Arroyo Burro watershed in Santa Barbara, CA is an example, with its popular, but chronically FIB-contaminated beach. To address, a microbial source tracking study was performed. Surface waters were sampled over 2 years, FIB were quantified, and DNA was analyzed for host-associated fecal markers. Surf zone FIB were only elevated when the coastal lagoon was discharging. Among the fecal sources into the lagoon, including upstream human sources and coastal birds, canines were the most important. Canine sources included input via upstream creek water, which decreased after creek-side residences were educated about proper pet waste disposal, and direct inputs to the lagoon and surf zone, where dog waste could have been tidally exchanged with the lagoon. Based on this study, canine waste can be an influential, yet controllable, fecal source to suburban coastal beaches.

Comparison of the Host Specificities of Two Bacteroidales Quantitative PCR Assays Used for Tracking Human Fecal Contamination

ABSTRACT The sewage-associated real-time quantitative PCR (qPCR) assays BacHum and HF183 SYBR were compared for specificity against local fecal sources. Both assays were equally sensitive to sewage, but BacHum showed substantially more false-positive results for cat, dog, gull, and raccoon feces.